The goal of the proposed research is to identify and characterize the role that the [2Fe- 2S] cluster of ferrochelatase, the terminal enzyme of heme biosynthesis, plays during erythroid heme synthesis. We have been stimulated to approach this question by recent discoveries with the zebrafish pinotage mutant, which is characterized by a profound hypochromic, microcytic anemia, but without accumulation of free protoporphyrin. Unpublished data have determined that the mutation causing this defect is in the gene encoding Atpif1, a protein not directly associated with heme biosynthesis, but one that does have an impact on the erythroid cell's ability to synthesize heme. This mutant phenotype is suppressed by replacement of the native ferrochelatase, which possesses a [2Fe-2S] cluster, with ferrochelatase from Saccharomyces cerevisiae, which lacks a [2Fe-2S] cluster. We hypothesize that the ferrochelatase [2Fe-2S] cluster modulates heme synthesis by sensing and responding to the mitochondrial pH and/or membrane potential that were found to be altered in the pinotage zebrafish. Such a postulated role is chemically and biologically feasible, and similar in some regards to the role played by the [2Fe-2S] cluster of the Escherichia coli transcriptional regulato SoxR. The specific aims of the proposal are to determine: 1) the impact of altered [2Fe-2S] cluster midpoint potential on in vivo ferrochelatase activity, 2) if the solvent-filled channel in which the [2Fe-2S] cluster resides is essential to the regulatory function of the cluster, 3) if th putative regulatory role of the cluster is unique to ferrochelatases of organisms that possess erythroid cells, and 4) if the [2Fe-2S] cluster exerts its function via an intra-protein action tha directly impacts catalysis and/or via inter-protein interactions between ferrochelatase and mitoferrin1 and/or protoporphyrinogen oxidase. For all ferrochelatase variants produced and utilized in these studies, kinetic and biophysical parameters will be determined and the structural integrity of the proteins validated by x-ray crystallography. The fact that pinotage mutants have diminished ferrochelatase activity, but do not accumulate free protoporphyrin as one finds in erythropoietic protoporphyria (EPP), indicates that loss of ferrochelatase activity pe se does not necessarily result in EPP, but in some circumstances can cause anemia. The study results will impact the field of erythropoiesis significantly since they will establish a new regulatory player in red cell heme synthesis. Additionally, new areas of investigation will open and the molecular basis of currently undefined red cell-based syndromes and diseases may be revealed.